1LMU Klinikum der Universität München Medizinische Klinik und Poliklinik I München, Deutschland; 2Gene and Cell Therapy Institute, Massachusetts General Brigham Boston, USA; 3Icahn School of Medicine at Mount Sinai New York, USA
Pulmonary arterial hypertension (PAH) is a disease characterized by dysregulated pulmonary vascular remodeling that leads to an increased pulmonary vascular resistance, uncompensated heart failure, and ultimately death. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, which regulate gene expression at the post-transcriptional level, thereby regulating cell growth, proliferation, migration, and differentiation. MiR-32 has been shown to be the most upregulated miR in lungs of patients with PAH and to promote the proliferation of liver cancer cells. These findings prompted further functional characterization of this miRNA in the setting of PAH. Our results indicate that miR-32 levels are increased in the lungs of PAH-diseased mice and pigs. We also found miR-32 inhibition in human pulmonary artery smooth muscle cells (PASMCs) and in pulmonary artery endothelial cells (PAECs) to confer an anti-proliferative phenotype in these cells in vitro. We next tested the therapeutic effect of miR-32 inhibition using the Hypoxia/Sugen model in mice. miR-32 inhibition was achieved via an intratracheal delivery of a chemically modified antisense oligonucleotide specific for miR-32 (LNA-32). LNA-Ctrl-treated mice displayed all the hallmarks of PAH (i.e., increased Fulton index, RVSP, cardiomyocyte hypertrophy and pulmonary arterial medial thickness), whereas LNA-32-treated mice displayed a marked and significant decrease in these parameters. In addition, we found that miR-32 targets several members of the adrenomedullin/cyclic adenosine monophosphate (ADM/cAMP) signaling pathway. To identify miR-32 targets, we transfected human pulmonary vascular cells with miR-32 or miR-Ctrl and performed RNA sequencing. By combining the RNA-seq data with the results of multiple target prediction programs and by performing an enrichment analysis of the identified genes using the Reactome pathway database, we found that miR-32 targets several members of the adrenomedullin/cyclic adenosine monophosphate (ADM/cAMP) signaling pathway. Our results indicate that miR-32 inhibition suppresses pulmonary vascular cell proliferation in vitro and pathological vascular remodeling in vivo by targeting the ADM-cAMP paracrine-signaling pathway.